PROJECT SUMMARY/ABSTRACT Sepsis is the leading cause of death in U.S. hospitals and is a medical emergency that requires immediate treatment. Every hour that treatment is delayed, sepsis-related mortality increases by ~8%, and ~80% of sepsis deaths can be prevented with rapid diagnosis and treatment. Hence, providing clinicians with a tool for rapid bacterial strain identification and drug susceptibility testing will enable timely and effective treatment, reduce the non-specific use of broad-spectrum antimicrobials, and improve clinical outcomes. Conventional assays for diagnosis of bloodstream infections rely on blood cultures, which are time- consuming and delay rapid and targeted treatment decisions. Molecular technologies that use bacterial nucleic acid amplification are faster than culture-based methods, but often require pre-analytic processing which adds time and complexity to the assay. Although some commercial assays are even more rapid, technologies that can further expedite bacterial identification and drug screening will impact current sepsis survival rates. The goal of this SBIR Phase I project is to demonstrate feasibility of rapid and sensitive bacterial capture, strain identification and drug susceptibility testing from whole blood. The system is based on our novel actuating surface-attached posts (ASAP) technology. ASAPs are an array of magnetic micro-posts that are flexible in the presence of a magnetic field. In this project, ASAP motion will expedite bacterial binding to an immobilized antibody microarray on the cartridge floor that consists of antibodies specific to several bacterial strains. The final device will have antibodies specific to the 20 most common bacterial strains that infect blood, with one antibody per bacterial strain. Antibodies will be arranged in 20 rows and 30 columns. Each column will contain all 20 antibodies whose positions in the 30 columns will be scrambled. The locations of all antibody spots on the microarray will be pre-determined. For this Phase I proof-of-concept study, antibodies to five bacterial strains will be printed on the cartridge floor. Following bacteria capture, the chamber will be washed and the captured bacterial strain(s) will be immediately (in 20 min) identified using a fluorescent mannose lectin binding (MBL) antibody that binds to MBL found on the surfaces of pathogens. Captured bacterial strain(s) will then be cultured in-cartridge, with ASAP motion circulating the culture media to optimize bacterial growth conditions and transport newly formed bacteria to adjacent antibody spots on the microarray floor. Drug solutions, each in three different concentrations (i.e., susceptible, intermediate and high), will be added to the bacteria and time-lapse imaging will evaluate bacterial growth to determine drug susceptibility. The significant advantage of our device is bacterial strain identification in whole blood in 20 min and drug screening in 2-4 hours, which are ~5x and at least 2x faster, respectively, than existing assays.